Isolation and Characterization of a Neutral Glycosphingolipid from the Epimastigote Form of Trypanosoma mega1

ABSTRACT. A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and α-hydroxy fatty acids. Normal C_16:0, C_18:0, and 2-hydroxy C_18:0 are the predominant fatty acids.     

Separation and Identification of Fatty Acids

An improved method is described for separating saturated fatty acids by reversed-phase paper chromatography. The 2,4-dinitrophenylhydrazides prepared from saturated fatty acids from C₂ to C₂₂ are run upward on paper impregnated with tetralin, using 90% methanol—acetic acid—tetralin, 80% ethanol—acetic acid—tetralin, or 90% methanol—tetralin as the moving solvent. The simultaneous separations of all even-carbon acids from C₆ to C₂₂, all odd-carbon acids from C₇ to C₁₉, and also all odd- and even-carbon acids from C₇ to C₁₉ can be successfully performed by means of this paper chromatography. The method is useful for the detection of component saturated fatty acids in natural fats.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Constituents of Fusel Oils Obtained Through the Fermentation of Corn,Barley and Sweet Molasses

The components of the fusel oils obtained through the fermentation of corn, barley and sweet molasses were separated by fractional distillation and adsorption chromatography. Each of the components was analyzed by gas chromatography and infrared spectrometry. Newly isolated components were as follows: methyl heptenone, fatty acids having odd number of carbon atoms, acetic esters of higher aliphatic alcohols ranging from C₆ to C₁₁, benzyl alcohol, ethyl phenylacetate, phenylethyl propionate, acetophenone, limonene, and α-ionone. Moreover, an unknown ketone C₁₀H₁₆O were isolated from corn fusel oil, and trans-neroridol from sweet molass fusel oil.     

Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography.

A rapid method is described for the analysis of mixtures of short-chain acyl coenzyme A thioesters by reversed-phase ion-pair chromatography on LiChrosorb RP-8 and μBondapak C₁₈ columns. The technique is applicable to separation of CoASH, acetyl-CoA propionyl-CoA, and 3-hydroxy-3-methylglutaryl-CoA, as well as dicarboxylic acids and several nucleotides commonly used as cofactors for biosynthetic reactions. The method was utilized on a preparative scale for purification of 3-hydroxy-3-ethylglutaryl-CoA from CoASH and 3-hydroxy-3-ethylglutaric acid. The counterion employed was tetrabutylammonium (phosphate), pH 5.5, in various methanol:water mixtures. Elution profiles and retention values of compounds were influenced by the concentration of counterion and mass of injected sample. Tetrabutylammonium ions could be removed from effluent by ionexchange chromatography on Amberlite IR-120 resin.     

The utilization of short-chain monocarboxylic acids as carbon sources for the production of gamma-linolenic acid by Mucor strains in fed-batch culture

The Fischer-Tropsch reaction water, which contains C₂ to C₅ monocarboxylic acids, generated as a co-product of the Sasol industrial oil-from-coal process, constitutes a potential cheap carbon substrate for the production of gamma-linolenic acid (GLA) by selced Mucor species. Three strains of Mucor were each grown in an air-lift reactor operated in a fed-batch, pH-stat mode under N-limitation with a mixture of C₂ to C₅ monocarboxylic acids as both pH titrant and carbon source. The production of GLA from this substrate was evaluated. Growth typically resulted in the rapid assimilation of acetic, n-butyric and n-valeric acids. Although propionic, iso-butyric and iso-valeric acids were assimilated to varying degrees, these acids accumulated in the culture. Mucor circinelloides CBS 203.28 gave the best results in that it assimilated 36% to 100% of each acid, had a biomass yield coefficient of 0.3 (calculated on acids utilized), and contained 28% crude oil, 84% of which comprised neutral lipids with a GLA content of 14.4%, giving 33 mg GLA/g biomass. GLA accumulation coincided with a decrease in the stearic-acid content of the neutral-lipid fraction. The results were comparable with previous results obtained with acetic acid and glucose as sole carbon sources, demonstrating the feasibility of producing GLA from the above mixture of organic acids.     

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C₈ preparative column with 6% acetonitrile—45% methanol—5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-μm Spin-X cartridges, applied to a reversed-phase C₁₈ column, and eluted in a 0–50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzylamiloride.     

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II³SO₃LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C₆ to C₈ fatty acids, C₆-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C₁₆ and C₁₈ fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.     

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs–Ringer bicarbonate buffer only, 0–1 mM [3,4-¹³C₂]-4-hydroxynonanoate, and 0–2 mM [5,6,7-¹³C₃]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-¹³C₃]heptanoate perfusates.     

Brain sphingoglycolipids in Krabbe's globoid cell leucodystrophy

Abstract— Seven sphingoglycolipids were isolated from the white matter of a patient with globoid cell leucodystrophy (Krabbe's disease). After purification by saponification and column and preparative thin-layer chromatography, these compounds were analysed for the carbohydrate composition and sequence and for fatty acid composition by paper and gas-liquid chromatography. The compounds were identified as gluco- and galactocerebrosides, lactosyl-ceramide, digalactosy I-glucosyl-ceramide, two types of tetrahexosyl-ceramides (asialo-ganglioside and globoside), and sulphatide. Glucocerebrosideconstituted 13 percent of total cerebroside in white matter, but sulphatide contained only galactose. Galactocere-broside and sulphatide exhibited compositions of fatty acids similar to those in normal white matter, with only minor abnormalities. Other sphingoglycolipids showed fatty acid patterns with relatively high proportions of longer-chain fatty acids, rather than the predominant C_18:0 acid usually found in ceramide hexosides of the brain. Hematoside, also found in the white matter in a significant amount, similarly contained a large proportion of longer-chain fatty acids, whereas other gangliosides contained predominantly C_18:0 acid. The abnormal ceramide hexoside pattern was restricted mostly to white matter except for glucocerebroside, which constituted 32 per cent of grey matter cerebroside. We postulate that the visceral type of sphingoglycolipids may be constituents of globoid cells, abundantly present in white matter and considered to be cells of mesenchymal origin.     

Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitrificans NG80-2

The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C₂) to melissic acid (C₃₀). The most preferred substrates were capric acid (C₁₀) for Facl1 and palmitic acid (C₁₆) for Facl2, respectively. Both enzymes had an optimal temperature of 60 °C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C₃₀ has been demonstrated in bacteria.     

Plant regeneration from mesophyll protoplasts of Moricandia arvensis

Moricandia arvensis is of interest as it is a dicotyledonous species which has C₃ — C₄ intermediate photosynthesis, a mechanism which results in enhanced recapture of photorespired CO₂. Leaves from cultured shoot tips were used as a source for mesophyll cell protoplasts. Approximately 1% of the protoplasts which survived the first few days of culture produced calli. On a suitable regeneration medium, 30–60% of the calli regenerated one or more shoots. From among the regenerating shoots eight were selected, transferred to soil and grown to flowering in the glasshouse; all were fertile. The development of a protoplast regeneration system provides the opportunity to use transformation and somaclonal variation as tools in the genetic analysis of the C₃–C₄ character in this species.Abbreviations GDC glycine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzyl aminopurine - NAA naphthalene acetic acid - ABA abscisic acid     

Simultaneous determination of hydroxocobalamin and its cyanide complex cyanocobalamin in human plasma by high-performance liquid chromatography application to pharmacokinetic studies after high-dose hydroxocobalamin as an antidote for severe cyanide poisoning

Hydroxocobalamin (OHCbl) is a powerful antidote for cyanide poisoning, via the formation of non-toxic cyanocobalamin (CNCbl). Plasmatic cobalamins were measured at 361 nm, after enrichment and purification on a short C₁₈ precolumn (1% acetic acid; 1 ml min⁻¹; 2 min), by back-flush elution on a C₁₈ ODS-2 column [0.1 M sodium dihydrogenphosphate-methanol (63:27, v/v) (pH 4.0); 0.80 ml min⁻¹]. The precision was 3.21 and 3.54% for 10 μM OHCbl and CNCbl, respectively. The method was used to study the pharmacokinetics of OHCbl and the formed CNCbl in severely poisoned patients.     

Effects of various organic acid supplements on growth rates of Eubacterium limosum B2 on methanol

Summary During anaerobic growth on methanol, Eubacterium limosum B2 produces acetic and butyric acids as overflow metabolites, but can be induced to produce other organic acids. All organic acids (C₂–C₆) tested had a similar effect on growth, although the toxicity of each was different e.g. increasing inhibition by acids of increasing chain length. Inhibition was only observed above a threshold concentration related to the molecular size of the organic acids. At higher concentrations the degree of inhibition was a linear function of concentration. In a mathematical treatment of the data the inhibition constant (K _p) was shown to be proportionate to the threshold value (P _c) of each organic acid and accurately predicted the growth characteristics of Eubacterium limosum on methanol following the addition of organic acid supplements.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Purification of growth hormones by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (HPLC) on a column of Radial-Pak C₁₈ cartridge was utilized for the purification of a variety of growth hormone (GH) proteins from mammalian, avian, amphibian and fish pituitary glands. Recovery of GH from pituitary glands of up to 0.43% of total protein was obtained with a high degree of homogeneity as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The HPLC-purified GHs show reactions of identity or near identity by immuno-diffusion studies on agar gel. This method offers a convenient and rapid purification of vertebrate GH on an analytical or preparative scale.     

Halogenolysis of para-substituted benzyl cobaloximes. Part II.

The reaction of benzyl cobaloximes with halogens (Cl₂ or Br₂) in chloroform or acetic acid forms benzyl halides and benzyl ethers of dimethylgly- oximes by an oxidative dealkylation mechanism.     

Venom and Dufour's gland secretions in an Australian species of Camponotus

Constituents of the venom (1) and Dufour's gland (25) have been characterized in an Australian representative of the highly evolved ant subfamily Formicinae. The venom reservoir of this ant, Camponotus intrepidus, contains formic acid, identified as the benzyl ester. The Dufour's gland contains a major hydrocarbon and a minor fatty acid fraction. Hydrocarbons include the normal alkanes, C₁₀ to C₁₇ (82 per cent); two series of monomethylalkanes, C₁₂, C₁₃, C₁₄, C₁₆, and C₁₇, the 3-methyl derivatives comprise approximately 16 per cent, and the 5-methylalkanes 2 per cent of the total; there are trace proportions of the n-alkenes, C₁₂, C₁₃, and C₁₅. The minor fatty acids, myristic, pentadecanoic, palmitic, and stearic are present in the ratio 2 : 2 : 12 : 11.     

Microbial Oxidation of Hydrocarbons and Related Compounds by Whole-Cell Suspensions of the Methane-Oxidizing Bacterium H-2

Previously, a thermophilic obligate methane-oxidizing bacterium, H-2 (type I), was isolated in our laboratory. H-2 is a new type of methylotroph because of the G+C content of DNA; it uses both the ribulose monophosphate pathway and the serine pathway for carbon assimilation and possesses a new quinone. In addition, we found that resting cell suspensions of H-2 had the ability to oxidize a variety of compounds different from the other methane-oxidizing bacteria as follows. (i) C₁ to C₈n-alkanes are hydroxylated and further oxidized, yielding mixtures of the corresponding alcohols, aldehydes, acids, and ketones. Liquid alkanes are transformed through a different oxidative pathway from that of gaseous ones. (ii) Both gaseous (C₂ to C₄) and liquid (C₅, C₆) n-alkenes are oxidized to their corresponding 1,2-epoxides. (iii) Liquid monochloro and dichloro n-alkanes (C₅, C₆) are oxidized, yielding their corresponding acids or haloacids. (iv) Diethyl ether is oxidized to acetic acid; no ethanol and acetaldehyde are detected. (v) Cyclic and aromatic compounds are also oxidized. (vi) Secondary alcohols (C₃ to C₁₀) are oxidized to their corresponding methyl ketones.